An hexa-nucleotide GGGGCC repeat expansion (HRE) was identified as the most common genetic cause of C9orf72-linked amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Both sense and antisense HRE strands of C9orf72 are translated by a repeat associated non-canonical AUG translation (RANT) mechanism in mutation carriers, resulting in the production of neurotoxic dipeptide- repeat proteins (DPRs). The mechanism and dynamics of RANT are largely unknown. For example, is RANT occurring only in disease-degenerating cells (e.g. cortical and motor neurons) versus other non- degenerating cell types (e.g., glia cells)? What are the molecular and cellular signals that initiate RANT? We hypothesize that the most important component for translation, canonical or non-canonical, is the choice for tRNA to read a specific codon to control the speed and quality of translation in specific cells and location. The critical barrier to answer these questions and to make progress in the field is the lack of experimental tractable models in disease?relevant cells. Here we propose: 1. To develop C9-ALS/FTD relevant cell-based models, using primary neurons and astrocytes as experimental platforms to study cell-specificity and molecular trigger of RANT driven by the C9orf72 HRE; 2. To develop a cell-free model system using brain ribosomes and tRNA pools to study the tRNA requirements for RANT driven by the C9orf72 HRE. These models will exploit distinct yet complementary aspects of C9orf72-linked RANT (herein referred as C9-RANT). A number of important questions will be addressed using these models: (i) Is the production of certain DPRs dependent on cell types? (ii) Do cellular stressors trigger or increase C9-RANT? (iii) Which tRNA isodecoders are used to read the Pro, Arg, Ala, Gly codons by RANT? The answers will define the basic parameters of C9-RANT and its pathogenic implication for C9-ALS/FTD. We believe we have unique and complementary expertise to successfully address these questions, which are not to the best of our knowledge being pursued elsewhere.